Inhibitor of differentiation (Id) proteins are transcriptional regulators implicated in diverse developmental, physiologic and pathologic processes such as cancer and atherogenesis, and the Id3 genes and proteins are an important member of this class [Lim et al., Acta Pharmacol Sin; 26:1409-20 (2005)].
Id genes are widely expressed in the animal kingdom from humans to zebra fish [Dickmeis et al., Mech Dev; 113:99-102 (2002)]. Four Id genes, Id1-Id4, have been identified in humans and in rodents. A homologous Id-like gene, extramacrochaetae has been identified in drosophila [Campuzano et al., Oncogene; 20:8299-307 (2001)]. The name, inhibitor of differentiation, derives from the property of the Id proteins to diminish the differentiation of a variety of cells by inhibiting the DNA binding activity of many transcription factors that regulate expression of cell-type specific genes.
The Id proteins are small proteins of approximately 13 kDa-20 kDa. All four Id proteins contain a modestly conserved helix-loop-helix (HLH) structural motif in the middle of the protein, but are otherwise divergent in sequences. The four Id proteins constitute one subclass (Class V) of the large family of HLH transcriptional regulators. Unlike other HLH proteins that can bind to DNA as homodimers or heterodimers, the Id proteins lack the basic amino acid domain needed for DNA binding. Instead, they function primarily by forming heterodimers with the “ubiquitous” Class I HLH proteins known as E-proteins. This dimerization prevents the E-proteins from interacting with each other and with cell-type specific Class II HLH proteins by inhibiting their binding to DNA and repressing their ability to modulate gene expression. This modulation can include both inhibition and stimulation of gene expression. For example, some class I bHLH proteins repress transcription and with these repressor proteins sequestration could ameliorate their inhibitory effects and lead to increased gene expression. For other transcription factors such as ETS, SREBP-1, Pax5, etc., Id proteins interact and inhibit. Certain individual members of the Id family proteins interact selectively with specific proteins. Individual Id proteins might interact selectively with proteins not recognized by other Id family members. For example, Id1 is the only Id protein shown to bind the proteasomal protein S5a [Anand et al., J Biol Chem; 272:19140-51 (1997)], and as previously mentioned only Id2 binds to the tumor suppressor retinoblastoma protein Rb and interferes with the ability of hypophosphorylated Rb to suppress cell proliferation when both are ectopically expressed [Lasorella et al., Mol Cell Biol; 16:2570-8 (1996)].
The Id family proteins have been extensively reviewed [Sidker et al., Cancer Cell; 3:525-30 (2003), Benezra et al., Oncogene; 20:8334-41 (2001), Lasorella et al., Oncogene; 20:8326-33 (2001), Yokota et al., J. Cell Physiol; 190:21-28 (2002), Ruzinova et al., Trends Cell Biol; 13:410-18 (2003)]. Most of these reviews have dealt with the Id proteins as a group and concentrated primarily on the potential biological functions of the Id proteins. Relatively less attention has been devoted to reviewing the molecular mechanisms that regulate the expression and function of individual Id genes and proteins.
The Id3 gene was first identified as a serum- inducible immediate early gene in an established murine fibroblastic cell line [Christy et al., Proc Natl Acad Sci USA; 88:1815-27 (1991)]. Subsequent studies have documented their involvement in various biological processes, including T and B cell development, skeletal muscle differentiation [Atherton et al., Cell Growth Differ; 7:1059-66 (1996), Melnikova et al., Cell Growth Differ; 7:1067-79 (1996)], vascular smooth muscle cell proliferation [Forrest et al., J Biol Chem; 279:32897-903 (2004), Deed et al., FEBS Lett; 393:113-6 (1996)], embryonic neurogenesis, osteogenesis [Maeda et al., J Cell Biochem; 93:337-44 (2004)], and tumor-induced angiogenesis. Expression and function of the protein is under many complex layers of regulation and, therefore, could provide rich targets for therapeutic interventions.
Several studies have characterized the expression of Id3 at either the mRNA or the protein level. A wide range of techniques have been utilized, including Northern, in situ hybridization, reverse transcription with polymerase chain reaction, various genome expression profiling assays, Western immunoblots and immunocytochemical staining procedures. Like other Id genes, the expression of Id3 is dynamically regulated during embryonic development. The general expression level is high at the early embryonic ages, but progressively declines as the embryo develops [Ellmeier et al., Dev Dyn; 203:163-73 (1995)]. Id3 is widely expressed throughout the embryo proper. Its expression is readily detectable within regions that are undergoing active morphogenesis [Jen et al., Dev Dyn; 207:235-52 (1996)], but can also be detected in some undifferentiated tissues.
Id3 is expressed by many, but not all, cells indicating that its regulation is likely to involve both ubiquitous as well as cell-type specific regulatory mechanisms. Perturbation of Id3 expression has been correlated with a variety of disease states and pathologic situations, including cancer, aging, atherosclerosis, muscle atrophy, and inflammation. Conversely, altered expression of Id3 has been detected during the regenerative process following tissue injury.
It is generally believed that members of the Id gene family behave like oncogenes. Overexpression of one or more Id genes has been detected in various cancers. The situation with Id3 is consistent in most part with this generalization [Wilson et al., Cancer Res; 61:8803-10 (2001), Langlands et al., Cancer Res; 60:5929-33 (2000)], but there are some exceptions. In certain neurological tumors, Id3 upregulation is observed not only in the tumors themselves but also in the vascular tissues surrounding the tumors [Vandeputte et al., Glia; 38:329-38 (2002)]. In contrast, expression is reduced in papillary thyroid carcinoma [Deleu et al., Exp Cell Res; 279:62-70 (2002)] and ovarian carcinomas [Arnold et al., Br J Cancer; 84:352-9 (2001)], and either increased [Sablitzky et al., Cell Growth Differ; 9:1015-24 (1998)] or absent [Albanese et al., Diagn Mol Pathol; 10:248-54 (2001)] in seminoma. The expression pattern is even more complex during the development of liver diseases and liver cancer. Id3 expression is low in normal liver, increases with the progression of liver diseases from chronic hepatitis to liver cirrhosis and is expressed at high levels in well-differentiated hepatocarcinomas, but not in the more advanced de-differentiated tumors [Damdinsuren et al., Int J Oncol; 26:319-27 (2005)].
Id3 expression level also changes in inflammatory and atherogenic processes. Id gene expression is upregulated in reactive astrocytes activated as part of the inflammatory process following spinal cord injury [Tzeng et al., Glia; 26:139-52 (1999)]. Id3 expression is also altered in vascular smooth muscle cells (VSMC) during atherogenesis. It is expressed at low level in normal vessels of the carotid artery, but is increased within 3 days of balloon injury and remains high through 14 days post injury. This is accompanied by the appearance of a novel differentially spliced Id3 transcript.
The inhibitor of differentiation transcription factor Id3 is implicated in numerous, diverse developmental, physiologic and pathophysiologic processes. Although some overlap with the other Id genes and protein is apparent, Id3 has a distinct expression, location and activity profile. Many of the biologic processes affected by Id3 are important to significant human disease such as cancer and atherogenesis as well as inappropriate immunity such as after tissue and/or organ transplantation. In addition, Id3 itself is regulated by many biologically important systems so that the amount of Id3 present in a particular matrix represents an integration of many important processes many of which are indicative of pathology. Methods to measure Id3 in biologic matrices to establish the Id3 status of the biologic test material are important for diagnostic, prognostic or research purposes. Such methods require an excellent sensitivity because Id3 is active at very low concentrations [Langlands et al., J Biol Chem; 272: 19785-93 (1997)]. Such methods also require a high degree of specificity with respect to other endogenous materials in the biologic matrices and other Id proteins.
Production of monoclonal antibodies specific to Id3 is difficult because of the relatively low molecular weight (about 17 kDa) and common structural homology among the Id proteins. Commercial rabbit polyclonal anti-mouse/human Id3 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) does not have desired properties because it has cross-reactivity with other proteins in a biological sample and it does not have high affinity to Id3. The undesired cross-reactivity and low binding affinity make the commercial antibodies unsuitable for use in immunoassays to detect Id3. It is difficult to prepare polyclonal antibodies specific only to human Id3, with no cross-reactivity to mouse Id3; or specific only to mouse Id3, with no cross-reactivity to human Id3 because of the homology between mouse Id3 and human Id3.
There is a need for antibodies specific for Id3, which do not substantially cross-react with other endogenous proteins in a biological sample or those within the Id family proteins such as Id1, Id2, and Id4. There is also a need for antibodies that have high binding constant to Id3 such that they are sensitive to detect or quantitate Id3 in biological samples. There is further a need for antibodies that are specific only to mouse Id3 or specific only to human Id3 with no substantial cross-reactivity between the two species, for research purpose.